Assembly of B4GALT1/ST6GAL1 heteromers in the Golgi membranes involves lateral interactions via highly charged surface domains.

ß-1,4-Galactosyltransferase 1 (B4GALT1) and ST6 ß-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) catalyze the successive addition of terminal ß-1,4-linked galactose and α-2,6-linked sialic acid to N-glycans. Their exclusive interaction in the Golgi compartment is a prerequisite for their full catalytic activity, whereas a lack of this interaction is associated with cancers and hypoxia. To date, no structural information exists that shows how glycosyltransferases functionally assemble with each other. Using molecular docking simulations to predict interaction surfaces, along with mutagenesis screens and high-throughput FRET analyses in live cells to validate these predictions, we show here that B4GALT1 and ST6GAL1 interact via highly charged noncatalytic surfaces, leaving the active sites exposed and accessible for donor and acceptor substrate binding. Moreover, we found that the assembly of ST6GAL1 homomers in the endoplasmic reticulum before ST6GAL1 activation in the Golgi utilizes the same noncatalytic surface, whereas B4GALT1 uses its active-site surface for assembly, which silences its catalytic activity. Last, we show that the homomeric and heteromeric B4GALT1/ST6GAL1 complexes can assemble laterally in the Golgi membranes without forming cross-cisternal contacts between enzyme molecules residing in the opposite membranes of each Golgi cisterna. Our results provide detailed mechanistic insights into the regulation of glycosyltransferase interactions, the transitions between B4GALT1 and ST6GAL1 homo- and heteromers in the Golgi, and cooperative B4GALT1/ST6GAL1 function in N-glycan synthesis.

A Golgi-associated redox switch regulates catalytic activation and cooperative functioning of ST6Gal-I with B4GalT-I.

Glycosylation, a common modification of cellular proteins and lipids, is often altered in diseases and pathophysiological states such as hypoxia, yet the underlying molecular causes remain poorly understood. By utilizing lectin microarray glycan profiling, Golgi pH and redox screens, we show here that hypoxia inhibits terminal sialylation of N- and O-linked glycans in a HIF- independent manner by lowering Golgi oxidative potential. This redox state change was accompanied by loss of two surface-exposed disulfide bonds in the catalytic domain of the α-2,6-sialyltransferase (ST6Gal-I) and its ability to functionally interact with B4GalT-I, an enzyme adding the preceding galactose to complex N-glycans. Mutagenesis of selected cysteine residues in ST6Gal-I mimicked these effects, and also rendered the enzyme inactive. Cells expressing the inactive mutant, but not those expressing the wild type ST6Gal-I, were able to proliferate and migrate normally, supporting the view that inactivation of the ST6Gal-I help cells to adapt to hypoxic environment. Structure comparisons revealed similar disulfide bonds also in ST3Gal-I, suggesting that this O-glycan and glycolipid modifying sialyltransferase is also sensitive to hypoxia and thereby contribute to attenuated sialylation of O-linked glycans in hypoxic cells. Collectively, these findings unveil a previously unknown redox switch in the Golgi apparatus that is responsible for the catalytic activation and cooperative functioning of ST6Gal-I with B4GalT-I.

One-Pot Multienzyme Cofactors Recycling (OPME-CR) System for Lactose and Non-natural Saccharide Conjugated Polyphenol Production.

A one-pot multienzyme cofactors recycling (OPME-CR) system was designed for the synthesis of UDP-α-d-galactose, which was combined with LgtB, a ß-(1,4) galactosyltransferase from Neisseria meningitidis, to modify various polyphenol glycosides. This system recycles one mole of ADP and one mole of UDP to regenerate one mole of UDP-α-d-galactose by consuming two moles of acetylphosphate and one mole of d-galactose in each cycle. The ATP additionally used to generate UDP from UMP was also recycled at the beginning of the reaction. The engineered cofactors recycling system with LgtB efficiently added a d-galactose unit to a variety of sugar units such as d-glucose, rutinose, and 2-deoxy-d-glucose. The temperature, pH, incubation time, and divalent metal ions for the OPME-CR system were optimized. The maximum number of UDP-α-d-galactose regeneration cycles (RCmax) was 18.24 by fed batch reaction. The engineered system generated natural and non-natural polyphenol saccharides efficiently and cost-effectively.

C1GALT1 predicts poor prognosis and is a potential therapeutic target in head and neck cancer.

Core 1 ß1,3-galactosyltransferase (C1GALT1) controls the crucial step of GalNAc-type O-glycosylation and is overexpressed in various human malignancies. However, its role in head and neck squamous cell carcinoma (HNSCC) remains unclear. Here we demonstrate that C1GALT1 expression is upregulated in HNSCC tumors and is associated with adverse clinicopathologic features. Moreover, high C1GALT1 expression predicts poor disease-free and overall survivals. C1GALT1 overexpression enhances HNSCC cell viability, migration, and invasion, which can be reversed by erlotinib. Silencing of C1GALT1 suppresses the malignant behavior both in vitro and in vivo. Mass spectrometry and lectin pull-down assays demonstrate that C1GALT1 modifies O-glycans on EGFR. Blocking O-glycan elongation on EGFR by C1GALT1 knockdown decreases EGF-EGFR binding affinity and inhibits EGFR signaling, thereby suppressing malignant phenotypes. Using molecular docking simulations, we identify itraconazole as a C1GALT1 inhibitor that directly binds C1GALT1 and promotes its proteasomal degradation, leading to significant blockade of C1GALT1-mediated effects in HNSCC cells in vitro and in vivo. Collectively, our findings demonstrate a critical role of O-glycosylation in HNSCC progression and highlight the therapeutic potential of targeting C1GALT1 in HNSCC treatment.

The Immunological Regulation Roles of Porcine ß-1, 4 Galactosyltransferase V (B4GALT5) in PRRSV Infection.

B4GALT5, also known as ß-1, 4 galactosyltransferase V, is one of the members of ß-1, 4 galactosyltransferase gene (B4GALT) family, which was concerned with embryonic development, tumor generation, other malignant diseases. In this study, we firstly cloned porcine B4GALT (pB4GALT5) from porcine alveolar macrophages, and predicted the structural domain and function of seven porcine ß-1, 4 galactosyltransferase (I-VII) based on transcriptome analysis of PRRSV infected cells. Additionally, the upregulated porcine B4GALT5 expression was detected from PRRSV infected porcine alveolar macrophage (PAM) cells. The PRRSV proliferation were slightly inhibited in overexpression of pB4GALT5 transfected cells, the interaction of B4GALT5 and GP5 of PRRSV was firstly be detected by Co-IP, and the co-location between B4GALT5 and GP5 were also observed in golgi membranes by confocal microscopy. A significant increasing mRNA transcription, including inflammatory cytokines (IFN-α, IL-6, IL-18, IL-1ß, TNF-α) and some cell surface glycosylated protein involved in antigen present (MHC-I/II), cell adhesion and migration (chemokine MCP-1 and receptor CCR2; LFA-1, ICAM-1) were upregulated in B4GALT5 overexpressed PRRSV infected cells. Our results demonstrated that the regulation of pB4GALT5 plays an important roles in PRRSV proliferation and modification function in viral infection cells. And these results will make achievements by supporting the research of latent mechanisms of ß-1, 4 galactosyltransferase V in antiviral immunity.

In Vitro and In Vivo Performance of Tissue-Engineered Tendons for Anterior Cruciate Ligament Reconstruction.

BACKGROUND: Anterior cruciate ligament (ACL) reconstruction is the current standard of care for ACL tears. However, the results are not consistently successful; autografts or allografts have certain disadvantages; and synthetic grafts have had poor clinical results. PURPOSE: To determine if recellularization of decellularized tendons combined with mechanical stimulation in a bioreactor could replicate the mechanical properties of the native ACL and be successfully used for ACL reconstruction in vivo. STUDY DESIGN: Controlled laboratory study. METHODS: Porcine tibialis tendons were decellularized and then recellularized with human adult bone marrow-derived stem cells. Tendons were cultured in a tissue bioreactor that provided biaxial cyclic loading for up to 7 days. To reproduce mechanical stresses similar to those experienced by the ACL within the knee joint, the tendons were subjected to simultaneous tension and torsion in the bioreactor. Expression of tendon-specific genes and newly synthesized collagen and glycosaminoglycan were used to quantify the efficacy of recellularization and dynamic bioreactor culture. The ultimate tensile load to failure and stiffness of recellularized constructs were measured after dynamic stimulation. Finally, the tissue-engineered tendons were used to reconstruct the ACL in 24 pigs, and ultimate tensile load to failure and stiffness were assessed after 3 months. RESULTS: Dynamic bioreactor culture significantly increased the expression of tendon-specific genes, the quantity of newly synthesized collagen and glycosaminoglycan, and the ultimate tensile load and stiffness of recellularized tendons. After in vivo reconstruction, the ultimate tensile load and stiffness of the tissue-engineered tendons increased significantly up to 3 months after surgery and were within 80% of the ultimate tensile load of the natural ACL. CONCLUSION: This translational study indicates that recellularization and dynamic mechanical stimuli can significantly enhance matrix synthesis and ultimate tensile load of decellularized porcine tibialis tendons. This approach to tissue engineering can be very useful for ACL reconstruction and may overcome some of the disadvantages of autografts and allografts. CLINICAL RELEVANCE: Dynamic bioreactor cultivation of tissue-engineered tendons may overcome the limitations of autografts and allografts.

A B3GALT6 variant in patient originally described as Al-Gazali syndrome and implicating the endoplasmic reticulum quality control in the mechanism of some ß3GalT6-pathy mutations.

Al-Gazali syndrome encompasses several clinical features including prenatal growth retardation, large joints contractures with camptodactyly, bilateral talipes equinovarus, small mouth, anterior segment anomalies of the eyes, and early lethality. Recently, a baby with features very similar to Al-Gazali syndrome was found to have compound heterozygous variants in B3GALT6. This gene encodes Beta-1,3-galactosyltransferase 6 (ß3GalT6), an essential component of the glycosaminoglycan synthesis pathway. Pathogenic variants in B3GALT6 have also been shown to cause Ehlers-Danlos syndrome spondylodysplastic type (spEDS-B3GALT6) and spondyloepimetaphyseal dysplasia with joint laxity type I (SEMD-JL1). In 2017, a new international classification of EDS included these 2 conditions together with the child reported to have features similar to Al-Gazali syndrome under spondylodysplastic EDS (spEDS). We report a disease-causing variant c.618C > G, p.(Cys206Trp) in 1 patient originally described as Al-Gazali syndrome and reported in 1999. We evaluated the involvement of the endoplasmic reticulum-associated protein degradation, in the pathogenesis of 13 B3GALT6 variants. Retention in endoplasmic reticulum was evident in 6 of them while the c.618C > G, p.(Cys206Trp) and the other 6 variants trafficked normally. Our findings confirm the involvement of B3GALT6 in the pathogenesis of Al-Gazali syndrome and suggest that Al-Gazali syndrome represents the severe end of the spectrum of the phenotypes caused by pathogenic variants in this gene.

'One-pot' sequential enzymatic modification of synthetic glycolipids in vesicle membranes.

ß(1,4)-Galactosyltransferase (ß4Gal-T1) and T. cruzi trans-sialidase (TcTS) have been used in a 'one-pot' cascade to provide vesicles (liposomes) with a trisaccharide coating. These soluble enzymes catalysed the transfer of galactose then sialic acid onto a synthetic N-acetylglucolipid embedded in the bilayers. Clustering of this substrate into microdomains increased the rate of sialylated lipid production, showing that an increase in ß4Gal-T1 activity is carried through the enzymatic cascade. These coatings modulated cell recognition. Hepatocellular carcinoma cells took up vesicles modified by ß4Gal-T1 alone more extensively than sialylated vesicles produced by 'one-pot' sequential enzymatic modification.

Enzymatic Synthesis of N-Acetyllactosamine (LacNAc) Type 1 Oligomers and Characterization as Multivalent Galectin Ligands.

Repeats of the disaccharide unit N-acetyllactosamine (LacNAc) occur as type 1 (Galß1, 3GlcNAc) and type 2 (Galß1, 4GlcNAc) glycosylation motifs on glycoproteins and glycolipids. The LacNAc motif acts as binding ligand for lectins and is involved in many biological recognition events. To the best of our knowledge, we present, for the first time, the synthesis of LacNAc type 1 oligomers using recombinant ß1,3-galactosyltransferase from Escherichia coli and ß1,3-N-acetylglucosaminyltranferase from Helicobacter pylori. Tetrasaccharide glycans presenting LacNAc type 1 repeats or LacNAc type 1 at the reducing or non-reducing end, respectively, were conjugated to bovine serum albumin as a protein scaffold by squarate linker chemistry. The resulting multivalent LacNAc type 1 presenting neo-glycoproteins were further studied for specific binding of the tumor-associated human galectin 3 (Gal-3) and its truncated counterpart Gal-3∆ in an enzyme-linked lectin assay (ELLA). We observed a significantly increased affinity of Gal-3∆ towards the multivalent neo-glycoprotein presenting LacNAc type 1 repeating units. This is the first evidence for differences in glycan selectivity of Gal-3∆ and Gal-3 and may be further utilized for tracing Gal-3∆ during tumor progression and therapy.

Characteristics of α-Gal epitope, anti-Gal antibody, α1,3 galactosyltransferase and its clinical exploitation (Review).

The α-Gal epitope (Galα1,3Galα1,4GlcNAc­R) is ubiquitously presented in non-primate mammals, marsupials and New World Monkeys, but it is absent in humans, apes and Old World monkeys. However, the anti-Gal antibody (~1% of immunoglobulins) is naturally generated in human, and is found as the immunoglobulin G (IgG), IgM and IgA isotypes. Owing to the specific binding of the anti­Gal antibody with the α­Gal epitope, humans have a distinct anti­α­gal reactivity, which is responsible for hyperacute rejection of organs transplanted from α­gal donors. In addition, the α1,3 galactosyltransferases (α1,3GT) can catalyze the synthesis of the α­Gal epitope. Therefore, the α1,3GT gene, which encodes the α1,3GT, is developed profoundly. The distributions of the α­Gal epitope and anti­Gal antibody, and the activation of α1,3GT, reveal that the enzyme of α1,3GT in ancestral primates is ineffective. Comparison of the nucleotide sequence of the human α1,3­GT pseudogene to the corresponding different species sequence, and according to the evolutionary tree of different species, the results of evolutionary inactivation of the α1,3GT gene in ancestral primates attribute to the mutations under a stronger selective pressure. However, on the basis of the structure, the mechanism and the specificity of the α­Gal epitope and anti­Gal antibody, they can be applied to clinical exploitation. Knocking out the α1,3GT gene will eliminate the xenoantigen, Gal(α1,3)Gal, so that the transplantation of α1,3GT gene knockout pig organ into human becomes a potential clinically acceptable treatment for solving the problem of organ shortage. By contrast, the α­Gal epitope expressed through the application of chemical, biochemical and genetic engineering can be exploited for the clinical use. Targeting anti­Gal­mediated autologous tumor vaccines, which express α­Gal epitope to antigen­presenting cells, would increase their immunogenicity and elicit an immune response, which will be potent enough to eradicate the residual tumor cells. For tumor vaccines, the way of increasing immunogenicity of certain viral vaccines, including flu vaccines and human immunodeficiency virus vaccines, can also be used in the elderly. Recently, α­Gal epitope nanoparticles have been applied to accelerate wound healing and further directions on regeneration of internally injured tissues.

QM/MM Studies Reveal How Substrate-Substrate and Enzyme-Substrate Interactions Modulate Retaining Glycosyltransferases Catalysis and Mechanism.

Glycosyltransferases (GTs) catalyze the biosynthesis of glycosidic linkages by transferring a monosaccharide from a nucleotide sugar donor to an acceptor substrate, and they do that with exquisite regio- and stereospecificity. Retaining GTs act with retention of the configuration at the anomeric carbon of the transferred sugar. Their chemical mechanism has been under debate for long as conclusive experimental data to confirm the mechanism have been elusive. In the past years, quantum mechanical/molecular mechanical (QM/MM) calculations have shed light on the mechanistic discussion. Here, we review the work carried out in our group investigating three of these retaining enzymes (LgtC, α3GalT, and GalNAc-T2). Our results support the controversial front-side attack mechanism as the general mechanism for most retaining GTs. The latest structural data are in agreement with these findings. QM/MM calculations have revealed how enzyme-substrate and substrate-substrate interactions modulate the transfer reaction catalyzed by these enzymes. Moreover, they provide an explanation on why in some cases a strong nucleophilic residue is found on the ß-face of the sugar, opening the door to a shift toward a double-displacement mechanism.

High Resolution Structures of the Human ABO(H) Blood Group Enzymes in Complex with Donor Analogs Reveal That the Enzymes Utilize Multiple Donor Conformations to Bind Substrates in a Stepwise Manner.

Homologous glycosyltransferases α-(1→3)-N-acetylgalactosaminyltransferase (GTA) and α-(1→3)-galactosyltransferase (GTB) catalyze the final step in ABO(H) blood group A and B antigen synthesis through sugar transfer from activated donor to the H antigen acceptor. These enzymes have a GT-A fold type with characteristic mobile polypeptide loops that cover the active site upon substrate binding and, despite intense investigation, many aspects of substrate specificity and catalysis remain unclear. The structures of GTA, GTB, and their chimeras have been determined to between 1.55 and 1.39 Å resolution in complex with natural donors UDP-Gal, UDP-Glc and, in an attempt to overcome one of the common problems associated with three-dimensional studies, the non-hydrolyzable donor analog UDP-phosphono-galactose (UDP-C-Gal). Whereas the uracil moieties of the donors are observed to maintain a constant location, the sugar moieties lie in four distinct conformations, varying from extended to the "tucked under" conformation associated with catalysis, each stabilized by different hydrogen bonding partners with the enzyme. Further, several structures show clear evidence that the donor sugar is disordered over two of the observed conformations and so provide evidence for stepwise insertion into the active site. Although the natural donors can both assume the tucked under conformation in complex with enzyme, UDP-C-Gal cannot. Whereas UDP-C-Gal was designed to be "isosteric" with natural donor, the small differences in structure imposed by changing the epimeric oxygen atom to carbon appear to render the enzyme incapable of binding the analog in the active conformation and so preclude its use as a substrate mimic in GTA and GTB.

Exploration of the active site of ß4GalT7: modifications of the aglycon of aromatic xylosides.

Proteoglycans (PGs) are macromolecules that consist of long linear polysaccharides, glycosaminoglycan (GAG) chains, covalently attached to a core protein by the carbohydrate xylose. The biosynthesis of GAG chains is initiated by xylosylation of the core protein followed by galactosylation by the galactosyltransferase ß4GalT7. Some ß-d-xylosides, such as 2-naphthyl ß-d-xylopyranoside, can induce GAG synthesis by serving as acceptor substrates for ß4GalT7 and by that also compete with the GAG synthesis on core proteins. Here we present structure-activity relationships for ß4GalT7 and xylosides with modifications of the aromatic aglycon, using enzymatic assays, cell studies, and molecular docking simulations. The results show that the aglycons reside on the outside of the active site of the enzyme and that quite bulky aglycons are accepted. By separating the aromatic aglycon from the xylose moiety by linkers, a trend towards increased galactosylation with increased linker length is observed. The galactosylation is influenced by the identity and position of substituents in the aromatic framework, and generally, only xylosides with ß-glycosidic linkages function as good substrates for ß4GalT7. We also show that the galactosylation ability of a xyloside is increased by replacing the anomeric oxygen with sulfur, but decreased by replacing it with carbon. Finally, we propose that reaction kinetics of galactosylation by ß4GalT7 is dependent on subtle differences in orientation of the xylose moiety.

Probing the acceptor active site organization of the human recombinant ß1,4-galactosyltransferase 7 and design of xyloside-based inhibitors.

Among glycosaminoglycan (GAG) biosynthetic enzymes, the human ß1,4-galactosyltransferase 7 (hß4GalT7) is characterized by its unique capacity to take over xyloside derivatives linked to a hydrophobic aglycone as substrates and/or inhibitors. This glycosyltransferase is thus a prime target for the development of regulators of GAG synthesis in therapeutics. Here, we report the structure-guided design of hß4GalT7 inhibitors. By combining molecular modeling, in vitro mutagenesis, and kinetic measurements, and in cellulo analysis of GAG anabolism and decorin glycosylation, we mapped the organization of the acceptor binding pocket, in complex with 4-methylumbelliferone-xylopyranoside as prototype substrate. We show that its organization is governed, on one side, by three tyrosine residues, Tyr(194), Tyr(196), and Tyr(199), which create a hydrophobic environment and provide stacking interactions with both xylopyranoside and aglycone rings. On the opposite side, a hydrogen-bond network is established between the charged amino acids Asp(228), Asp(229), and Arg(226), and the hydroxyl groups of xylose. We identified two key structural features, i.e. the strategic position of Tyr(194) forming stacking interactions with the aglycone, and the hydrogen bond between the His(195) nitrogen backbone and the carbonyl group of the coumarinyl molecule to develop a tight binder of hß4GalT7. This led to the synthesis of 4-deoxy-4-fluoroxylose linked to 4-methylumbelliferone that inhibited hß4GalT7 activity in vitro with a Ki 10 times lower than the Km value and efficiently impaired GAG synthesis in a cell assay. This study provides a valuable probe for the investigation of GAG biology and opens avenues toward the development of bioactive compounds to correct GAG synthesis disorders implicated in different types of malignancies.

Theoretical study of enzymatic catalysis explains why the trapped covalent intermediate in the E303C mutant of glycosyltransferase GTB was not detected in the wild-type enzyme.

Hybrid quantum mechanics/molecular mechanics calculations were used to study the catalytic mechanism of the retaining human α-(1,3)-galactosyltransferase (GTBWT) and its E303C mutant (GTBE303C). Both backside (via covalent glycosyl-enzyme intermediate, CGEI) and frontside SNi-like mechanisms (via oxocarbenium-ion intermediate, OCII) were investigated. The calculations suggest that both mechanisms are feasible in the enzymatic catalysis. The nucleophilic attack of the acceptor substrate to the anomeric carbon of OCII is the rate-determining step with an overall reaction barrier (ΔE(‡) = 19.5 kcal mol(-1)) in agreement with an experimental rate constant (kcat = 5.1 s(-1)). A calculated α-secondary kinetic isotope effect (α-KIE) of 1.27 (GTBWT) and 1.26 (GTBE303C) predicts dissociative character of the transition state in agreement with experimentally measured α-KIE of other retaining glycosyltransferases. Remarkably, stable CGEI in GTBE303C compared with its counterpart in GTBWT may explain why the CGEI has been detected by mass spectrometry only in GTBE303C ( Soya N, Fang Y, Palcic MM, Klassen JS. 2011. Trapping and characterization of covalent intermediates of mutant retaining glycosyltransferases. Glycobiology, 21: 547-552).

First functional analysis of a novel splicing mutation in the B3GALTL gene by an ex vivo approach in Tunisian patients with typical Peters plus syndrome.

Peters plus syndrome is a rare recessive autosomal disorder comprising ocular anterior segment dysgenesis, short stature, hand abnormalities and distinctive facial features. It was related only to mutations in the B3GALTL gene in the 13q12.3 region. In this study, we undertook the first functional analysis of a novel c.597-2 A>G splicing mutation within the B3GALTL gene using an ex-vivo approach. The results showed a complete skipping of exon 8 in the B3GALTL cDNA, which altered the open reading frame of the mutant transcript and generated a PTC within exon 9. This finding potentially elicits the nonsense mRNA to degradation by NMD (nonsense-mediated mRNA decay). The theoretical consequences of splice site mutations, predicted with the bioinformatics tool Human Splice Finder, were investigated and evaluated in relation to ex-vivo results. The findings confirmed the key role played by the B3GALTL gene in typical Peters-plus syndromes and the utility of mRNA analysis to understand the primary impacts of this mutation and the phenotype of the disease.

[Synthesis and acceptor properties of 11-[(9'-anthracenyl)methoxy]undecyl phosphate and P1-{11-[(9'-anthracenyl)methoxy]undecyl}-P2-(alpha-D-galactopyranosyl) diphosphate in the enzymic reactions catalyzed by galactosylphosphotransferase and mannosyltransferase from Salmonella newport].

Fluorescent 11-[(9'-anthracenyl)methoxy]undecyl phosphate and P1-{11-[(9'-anthracenyl)methoxy]undecyl}-P2-(alpha-D-galactopyranosyl) diphosphate were chemically synthesized for the first time. The ability of the first compound to serve as substrate-acceptor ofgalactosyl phosphate residue and the second compound of mannosyl residue in enzymic reactions catalyzed by galactosylphosphotransferase and mannosyltransferase from Salmonella newport membrane preparation was demonstrated.

In Porphyromonas gingivalis VimF is involved in gingipain maturation through the transfer of galactose.

Previously, we have reported that gingipain activity in Porphyromonas gingivalis, the major causative agent in adult periodontitis, is post-translationally regulated by the unique Vim proteins including VimF, a putative glycosyltransferase. To further characterize VimF, an isogenic mutant defective in this gene in a different P. gingivalis genetic background was evaluated. In addition, the recombinant VimF protein was used to further confirm its glycosyltransferase function. The vimF-defective mutant (FLL476) in the P. gingivalis ATCC 33277 genetic background showed a phenotype similar to that of the vimF-defective mutant (FLL95) in the P. gingivalis W83 genetic background. While hemagglutination was not detected and autoaggregation was reduced, biofilm formation was increased in FLL476. HeLa cells incubated with P. gingivalis FLL95 and FLL476 showed a 45% decrease in their invasive capacity. Antibodies raised against the recombinant VimF protein in E. coli immunoreacted only with the deglycosylated native VimF protein from P. gingivalis. In vitro glycosyltransferase activity for rVimF was observed using UDP-galactose and N-acetylglucosamine as donor and acceptor substrates, respectively. In the presence of rVimF and UDP-galactose, a 60 kDa protein from the extracellular fraction of FLL95 which was identified by mass spectrometry as Rgp gingipain, immunoreacted with the glycan specific mAb 1B5 antibody. Taken together, these results suggest the VimF glycoprotein is a galactosyltransferase that may be specific for gingipain glycosylation. Moreover, galatose is vital for the growing glycan chain.

Functional identification of a hydroxyproline-o-galactosyltransferase specific for arabinogalactan protein biosynthesis in Arabidopsis.

Although plants contain substantial amounts of arabinogalactan proteins (AGPs), the enzymes responsible for AGP glycosylation are largely unknown. Bioinformatics indicated that AGP galactosyltransferases (GALTs) are members of the carbohydrate-active enzyme glycosyltransferase (GT) 31 family (CAZy GT31) involved in N- and O-glycosylation. Six Arabidopsis GT31 members were expressed in Pichia pastoris and tested for enzyme activity. The At4g21060 gene (named AtGALT2) was found to encode activity for adding galactose (Gal) to hydroxyproline (Hyp) in AGP protein backbones. AtGALT2 specifically catalyzed incorporation of [(14)C]Gal from UDP-[(14)C]Gal to Hyp of model substrate acceptors having AGP peptide sequences, consisting of non-contiguous Hyp residues, such as (Ala-Hyp) repetitive units exemplified by chemically synthesized (AO)7 and anhydrous hydrogen fluoride-deglycosylated d(AO)51. Microsomal preparations from Pichia cells expressing AtGALT2 incorporated [(14)C]Gal to (AO)7, and the resulting product co-eluted with (AO)7 by reverse-phase HPLC. Acid hydrolysis of the [(14)C]Gal-(AO)7 product released (14)C-radiolabel as Gal only. Base hydrolysis of the [(14)C]Gal-(AO)7 product released a (14)C-radiolabeled fragment that co-eluted with a Hyp-Gal standard after high performance anion-exchange chromatography fractionation. AtGALT2 is specific for AGPs because substrates lacking AGP peptide sequences did not act as acceptors. Moreover, AtGALT2 uses only UDP-Gal as the substrate donor and requires Mg(2+) or Mn(2+) for high activity. Additional support that AtGALT2 encodes an AGP GALT was provided by two allelic AtGALT2 knock-out mutants, which demonstrated lower GALT activities and reductions in ß-Yariv-precipitated AGPs compared with wild type plants. Confocal microscopic analysis of fluorescently tagged AtGALT2 in tobacco epidermal cells indicated that AtGALT2 is probably localized in the endomembrane system consistent with its function.